Pathogens constantly threaten human health, and the problem is worsening; drug resistance by prevalent or emerging agents is growing, as is the potential for weaponization of familiar microbes by bioterrorists. Slowed development of antimicrobial agents has exacerbated the magnitude of this challenge. However, innovations in high-throughput screening (HTS) have made the path to compound discovery more systematic, promising both drug prototypes and research probes for gaining key insights into the molecular basis of infectious disease. Membranes are the first zones of combat between host and invading pathogens, making membrane-resident proteins central players in pathogenesis and defense. Over the past decade, proteases with active sites immersed inside the membrane have emerged at the core of circuits that diverse pathogens use to achieve virulence. Although these widespread microbial enzymes are now considered prime targets for combating infectious disease and/or drug resistance, potent inhibitors have never been isolated for microbial intramembrane proteases. Until now, these reactions have been inaccessible to direct screening in their natural membrane environment. We overcome this challenge by developing a system that allows controlling and monitoring non-invasively and in real-time rhomboid catalysis immersed inside the membrane. In response to NIH request PA-10-213, recently reissued as PA-13-364, we propose to use this innovation to develop novel HTS methods with these enzymes for the first time in their natural membrane setting, and to evaluate early hits emerging from our pilot screens. Successful outcomes raise the exciting possibility of ultimately contributing to the NIH's therapeutics for rare or neglected diseases (TRND) initiative. Methods pioneered in this proposal should be applicable to other membrane-immersed enzymes.